Retroviral Vector-Mediated Gene Transfer Applied For Studying The Mechanism Of Multidrug Resistance In Leukemia Cells

1. The Clinical Significance of MDR1, MRP, Bcl-2 Gene Expressionin Acute LeukemiaBy using the HL60/VCR, a human leukemia cell line with a typical MDR phenotype and high expression of P-glycorprotein, as a reference, MDR1 gene expression was determined in 9 tumor and leukemia cell lines and 54 cases with acute leukemia by using DIG-11-dUTP labeled RT-PCR assay. An objective and quantitative criterion were established for the assessment of MDR1 gene expression. By using conventional RT-PCR assay, MRP and Bcl-2 gene expression were also determined in same patients at same time. The results showed the positive rate of coexpression of MDRl/MRP/Bcl-2 in 54 leukemia patients was 16.7% (9/54) while 46.3% (25/54) of patients expressed none of these genes. The complete remission (CR) rate in patients who has no expression of either MDR1 or MRP, Bcl-2 gene was 80%, while no patients with a coexpression of MDRl/MRP/Bcl-2 or MDR1/MRP has achieved CR (PO.005). In terms of single gene expression in these leukemia patients the positive rate for MDR1, MRP or Bcl-2 was 28.3%, 41.3% and 47.8% respectively. Among them 15.4% of MDR1 positive cases achieved CR which was markedly lower than 72.7% in MDR1 negative cases (PO.005). 26.3% of MRP postive casesachieved CR which was markedly lower than 77.8% in MRP negative cases (PO.005). 36.4% of Bcl-2 postive cases achieved CR which was also significantly lower than 75.0% in negative cases (P<0.05). The patients with a coexpression of MDR1/MRP/ Bcl-2 or MDR1/MRP are extremely difficult to achieve CR. These results demonstrated a close relationship in acute leukemia patients between the drug-resistance phenotype and the expression of not only MDRlgene but also MRP and Bcl-2 gene.2. The Characteristics of Multidrug Resistance of the MDR1 GeneModified Leukemia Cell K562/MDRK562/MDR, a gene modified human leukemia cell line, was obtained by transduction of K562 cells with HaMDR retro viral vector who carries the full-lenth of human MDR1 gene cDNA,. By using PCR or RT-PCR analysis, we confirmed the integration and expression of MDR1 gene in K562/MDR cells. Moreover, K562/MDR cells expressed high level of P-gp determined by flow cytometry (FCM), indicating the MDR1 transgene can be effectively translated into P-gp, while the expression of atypical drug resistance protein MRP, LRP, BCRP and GST- n in K562/MDR cells had no change in comparison with the parental cell line K562. MTT assay has further revealed the resistance of K562/MDR to multiple anticancer agents. In addition, the sensitivity of K562/MDR to VCR can be completely restored by CsA and CRE either individually or in synergetic combination. These results showed the MDR phenotype of K562/MDR cells is solely mediated by MDRl/P-gp, per,se, but not other drug-resistant related genes. Thus, this gene modified drug-resistant cell line has provided a simple and sensitive in vitro model system for the study on the reversal of multidrug resistant in leukemia cells.3. Antisense MDR1 Gene Transfer to K562/MDR cells Mediated byRetrovirus vectorA 524 bp cDNA fragment of MDR1 containing the translation start codon ATGwas amplified from drug-resistant cell line HL60/VCR by high-fidelity RT-PCR. The recombinant retroviral vector carrying antisense MDR1 gene was then constructed into pLXSN by using conventional gene cloning techniques. The converted orientation of the MDR1 cDNA fragment was checked by appropriate restriction enzyme digestion. Both ecotropic and amphotropic retroviral producer cells were developed by liposome-mediated transfection and cross infection. The K562/MDR cells were then transduced with the supernatant of these producer cells and a successful integration of the antisense MDR1 cDNA and effectively expression of MDR1 antisense RNA were confirmed by PCR and RT-PCR assay. No helper virus could be found by both nested PCR and rescue assay. The efficiency and safety of the antisense gene transfer system may provide an experimental model in mutidrug resistance reversal experiment in cancer cel...